Methods and compositions for treating atheroma, tumors and other neoplastic tissues

ABSTRACT

Compositions comprising motexafin gadolinium, as well as methods for using these compositions in the treatment of brain metastases, are described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 10/950,302,filed Sep. 23, 2004, now U.S. Pat. No. 7,109,188 which is a divisionalapplication of U.S. patent application Ser. No. 09/699,027, filed Oct.27, 2000 and issued as U.S. Pat. No. 6,825,186 on Nov. 30, 2004, whichis a continuation-in-part of then co-pending U.S. patent applicationSer. No. 09/430,505, filed Oct. 29, 1999, which was converted toProvisional U.S. Patent Application No. 60/287,588, filed Oct. 29, 1999now abandoned, all of which are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel methods and pharmaceutical formulationsfor treating atheroma, tumors and other neoplastic tissue, as well asother conditions that are responsive to the induction of targetedoxidative stress. This invention also relates to novel methods fordetermining the radiation sensitization potential of a compound.

2. Publications Cited by Reference

Certain publications are cited in this application through the use ofthe following superscript numbers:

¹ Buettner, et al., Ridiation Research, Catalytic Metals, Ascorbate andFree Radicals: Combinations to Avoid, 145:532-541 (1996)

² Isoda, et al., J. Cancer Research, Change in Ascrobate RadicalProduction in an Irradiated Experimental Tumor with Increased TumorSize, 56:5741-5744 (1996)

³ Riley, Int. J. Radiat. Biol., Free Radical in biology: oxidativestress and the effects of ionizing radiation, 65(1):27-33 (1994)

⁴ Sessler, et al., J. Phys. Chem. A. One-Electron Reduction andOxidation Studies of the Radiations Sensitizer Gadolinium (III)Texaphyrin (PCI-120) and Other Water Soluble Metallotexaphyrins, 103:787-794 (1999)

⁵ Adams, et al., Radiation Res., 67:9-20 (1976)

⁶ Riley, Int. J. Radiat. Biol., Free Radicals in Biology: OxidativeStress and the Effects of Ionizing Radiation, 65(1):27-33 (1994)

⁷ Magda, et al., U.S. Pat. No. 5,798,491, Multi-Mechanistic ChemicalCleavage Using Certain Metal Complexes, issued Aug. 25, 1999

⁸ Young, et al., U.S. Pat. No. 5,776,925, Methods for CancerChemosensitization, issued Jul. 7, 1998

⁹ Sessler, et al., U.S. Pat. No. 5,622,946, Radiation SensitizationUsing Texaphyrins, issued Apr. 22, 1997

¹⁰ Sessler, et al., U.S. Pat. No. 5,457,183, Hydroxylated Texaphyrins,issued Oct. 10, 1995

¹¹ Sessler, et al., Accounts of Chem. Res., Texaphyrins: Synthesis andApplications, 27:43-50 (1994)

¹² Hemmi, et al., U.S. Pat. No. 5,599,928, Texaphyrin Compounds HavingImproved Functionalization, issued Feb. 4, 1997

¹³ Young, et al., Investigative Radiology, 29:330-338 (1994)

¹⁴ Mosmann, J. Immunol. Methods, 65:55-63 (1983)

¹⁵ Lin, et al., Analytical Biochemistry, The Cytotoxic Activity ofHematoheme: Evidence for Two Different Mechanisms, 161:323-331 (1987)

¹⁶ Volpin, et al., WO97/03666, EP 0 786 253 A1, U.S. Pat. No. 6,004,953,Agent for Suppressing Tumor Growth

All of the above publications are herein incorporated by reference intheir entirety to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referencein its entirety.

3. Background Information

The treatment of solid mammalian tumors with ionizing radiation involvesthe in situ generation of hydroxyl radicals and other reactive oxygenspecies which, due to the focusability of the ionizing radiation areprimarily located in the tumor, i.e., in tumor cells. These reactivespecies possess extreme oxidizing properties which oxidize biomoleculesin vivo thereby interfering with cellular metabolism.¹ For example, itis reported that ionizing radiation, such as X-rays and γ-rays, inducesirreversible damage to cellular DNA through production of hydroxylradicals and other reactive oxygen species in the cell leading to celldeath^(2,3) or initiation of the mechanism of apoptosis.⁴

One generally accepted mechanism of the cellular effect of ionizingradiation is initial damage inflicted to the cell's DNA by reactiveoxygen species generated by the ionizing radiation. In the presence ofmolecular oxygen, this damage is largely irreparable. Contrarily, in theabsence of molecular oxygen (such as hypoxic cells), cellularantioxidants such as ascorbate and NAD(P)H can act to repair damage tothe tumor DNA.

Tumor treatment via the use of ionizing radiation can be enhanced byincreasing the radiosensitivity of the tumor cells. One method suggestedfor enhancing radiosensitivity has been the external administration of acompound having a high affinity for electrons, which ideally localizesin the tumor. Proposed radiation sensitizers include compounds such ashalogenated pyrimidines, nitroimidazoles and gadolinium (III) complexesof the pentadentate macrocycle texaphyrin.⁴ Motexafon gadolinium (agadolinium (III) texaphyrin complex) is currently in Phase III clinicaltrials for the treatment of brain metastases.⁴

Phthalocyanine and naphthalocyanine polydentate ligands of thetransition metals cobalt and iron have been described as suppressing thegrowth of tumor cells when administered in combination with a biogenicreductant such as ascorbic acid.¹⁶

The observation that radiation sensitization occurs as a function ofredox potential gave rise to the proposal that such compounds functionby interception of aqueous electrons, thus preventing theirrecombination with cytotoxic radicals.⁵ Subsequent evidence showing alack of radiation sensitization activity for lutetium (III) texaphyrinin animal models notwithstanding the rapidity of reaction between thiscomplex and hydroxyl radicals under pulsed radiolytic conditions andminimal apparent nuclear localization suggest that this proposal mightnot fully explain the mechanism by which the gadolinium texaphyrins actas radiosenstizers.⁴

In view of the above, an understanding of the mechanism forradiosentization of tumor cells would be particularly helpful. Such anunderstanding could be used for testing in the discovery of newcompounds useful as radiation sensitizers as well as in maximizing thetherapeutic effect achieved by use of such compounds in the presence orthe absense of ionizing radiation.

SUMMARY OF THE INVENTION

This invention is premised upon the unexpected observation that theknown motexafin gadolium radiation sensitizer acts to catalyze theoxidation of NAD(P)H, ascorbate and other reducing agents underapproximate physiological conditions, leading to reactive oxygen speciesgeneration. Depletion of these reducing agents will inhibit biochemicalpathways that in vivo utilize reducing agents to effect repair of thedamage inflicted by reactive oxygen species. Additionally, sincehydrogen peroxide is recognized as probably the most significant of thereactive oxygen species⁶, the generation of hydrogen peroxide willfacilitate oxidative attack on the tumor or other tissue where it isproduced.

Moreover, this discovery that motexafin gadolium acts to catalyze theoxidation of reducing agents under approximate physiological conditionsto produce one or more reactive, oxygen species such as superoxide andhydrogen peroxide, serves as a basis to assess the radiation sensitizingpotential of other compounds. Specifically, a candidate compound can bescreened to determine its ability to generate one or more reactiveoxygen species under appropriate conditions. In turn, the amount ofreactive oxygen species so produced is then correlated to the radiationsensitizing ability of the compound. Accordingly, in one of its methodaspects, this invention is directed to a method for determining theradiation sensitization potential of a compound by the steps of:

a) introducing a compound to be tested into an aqueous solution of acellular metabolite having a standard biochemical reduction potentialmore negative than the standard biochemical reduction of oxygen/hydrogenperoxide;

b) monitoring the solution for the occurrence of a reaction thatproduces one or more reactive oxygen species; and

c) determining whether the compound has potential radiationsensitization activity, wherein the potential for radiationsensitization activity correlates to the occurrence of a reaction thatproduces one or more reactive oxygen species.

The reaction that produces a reactive oxygen species can be monitored innumerous manners as is well known to the skilled artisan, by measuringone or more of: depletion of oxygen, production of hydrogen peroxide,decreased concentration of the cellular metabolite, or generation of anoxidation product of the cellular metabolite. The cellular metabolite ispreferably a compound selected from the group consisting of ascorbate,NADPH, NADH, FADH₂ and reduced glutathione. Even more preferably, thecellular metabolite is ascorbate or NADPH.

In another embodiment, a compound which is determined to have radiationsensitization potential by a method of the present invention can beadministered to a mammalian host bearing a tumor and the tumor issubsequently exposed to ionizing radiation.

In still another embodiment there is provided a method for killing atumor cells by:

a) administering to the tumor cell a compound (other than a texaphyrin)that catalyzes the production of one or more reactive oxygen speciesfrom a cellular metabolite having a standard biochemical reductionpotential more negative than the standard biochemical reduction ofoxygen/hydrogen peroxide; and

b) exposing the cell to ionizing radiation. Preferably, the compoundpreferentially accumulates in tumor cells, e.g., as do porphyrinderivatives. One preferred compound is Fe(III)porphyrin.

The knowledge that certain compounds exhibiting radiation sensitizingproperties can catalytically effect the production of one or morereactive oxygen species from cellular metabolites having a biochemicalreduction potential more negative than oxygen/hydrogen peroxide alsoserves as a basis for a method to enhance the rate of tumor cell deathby co-administration of a thiol-depleting compound to the cell. Such athiol-depleting compound will reduce the level of thiol reducing agentssuch as glutathione thereby removing essential components of themetabolic pathways for repairing the cellular damage generated by thereactive oxygen species.

Accordingly, in another of its method aspects, this invention isdirected to a method for killing a tumor cell, which method comprises:

a) selecting a compound having radiation sensitization potential as perabove;

b) administering said compound to the tumor cell; and

c) co-administering to the tumor cell a thiol-depleting agent.

Preferably, the radiation sensitizing compound selected in step a) aboveis a texaphyrin and the thiol-depleting agent is buthionine sulfoximine.

The above method has applicability in the treatment of cancer in apatient. When so applied, this invention provides for a method oftreatment of cancer comprising administering to a patient sufferingtherewith an effective amount of a texaphyrin radiation sensitzer, aneffective amount of a thiol-depleting agent, and an effective amount ofionizing radiation.

In yet another of its method aspects, this invention is directed to amethod for killing a tumor cell which method comprises:

a) administering to said cell a compound that catalyzes the productionof one or more reactive oxygen species from a cellular metabolite havinga standard biochemical reduction potential more negative than thestandard biochemical reduction of oxygen/hydrogen peroxide; and

b) co-administering to said cell a second agent selected from the groupconsisting of DNA alkylators, topoisomerase inhibitors, redox cyclingagents, thiol-depleting agents, metabolic inhibitors, and mitochondrialinhibitors. Preferred DNA alkylators include carmustine. Preferred redoxcycling agents include alloxan, phenazine methosulfate, menadione,copper/putrescine/pyridine, methylene blue, paraquat, doxorubicin,bleomycin, and ruthenium (II) tris-(1,10-phenanthroline-5,6-dione).Preferred thiol-depleting agents include buthionine sulfoximine anddiethyl maleate. Preferred metabolic inhibitors include folic acidanalogs (e.g., methotrexate and trimetrexate), pyrimidine analogs (e.g.,fluorouracil, floxuridine, cytarabine and azacitidine), and purineanalogs and related inhibitors (e.g., mercaptopurine, thioguanine,pentostatin and fludarabine). Preferred mitochondrial inhibitors includeoligomycin and antimycin A. In another preferred embodiment, this methodfor killing tumor cells includes the additional step of exposing thecell to ionizing radiation.

In another, but related aspect of the invention, a first agent selectedfrom the group consisting of DNA alkylators, topoisomerase inhibitors,redox cycling agents, thiol-depleting agents, metabolic inhibitors, andmitochondrial inhibitors is co-administered with a second agent thatcatalyzes the production of one or more reactive oxygen species from acellular metabolite having a standard biochemical reduction potentialmore negative than the standard biochemical reduction of oxygen/hydrogenperoxide, to a subject having a condition (other than a tumor oratheroma) typically treated with such first agent.

Still further, in another aspect of this invention, there is provided amethod of selectively killing cells in a mammalian host bearing a tumoror atheroma, which method comprises:

a) administering to said mammalian host an agent that catalyzes theproduction of one or more reactive oxygen species from an intracellularreducing agent, preferably ascorbate or NAD(P)H;

b) optionally, allowing sufficient time for said agent to preferentiallyaccumulate in the cells of the tumor or atheroma; and

c) administering to said mammalian host a source or precursor of thereducing agent such as to increase the reactive oxygen speciesproduction in the tumor or atheroma.

In one preferred embodiment, this method further includes the steps ofexposing the tumor or atheroma to ionizing radiation. In anotherpreferred embodiment, the agent employed in this method is motexafingadolium or motexafin lutetium or combinations thereof.

In yet another aspect of this invention, there is provided a method oftreating a mammalian host bearing a tumor or an atheroma comprisingadministering to that host a therapeutically effective amount of acombination of motexafin gadolinium and motexafin lutetium and exposingthe tumor or atheroma to ionizing radiation.

In one of its composition aspects, this invention is directed topharmaceutical compositions for selectively killing cells in a hostbearing a tumor or atheroma comprising a pharmaceutically acceptablecarrier and an effective amount of an agent that catalyzes theproduction of one or more reactive oxygen species from an intracellularreducing agent provided that said agent is not a texaphyrin.

In another of its composition aspects, the invention encompasses anagent that preferentially accumulates in tumor or atheroma cells andcatalyzes the production of one or more reactive oxygen species from acellular metabolite having a standard biochemical reduction potentialmore negative than the standard biochemical reduction of oxygen/hydrogenperoxide, a source or precursor of the cellular metabolite, and apharmaceutically acceptable excipient.

Still further, this invention provides for use of an agent thatcatalyzes the production of one or more reactive oxygen species from anintracellular reducing agent in the treatment of mammalian tumors oratheroma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-6 illustrate the rate of NADPH oxidation versus NADPHconcentration by various metal cation complexes of a texaphyrin.

FIG. 7 illustrates the rate of ascorbate oxidation versus ascorbateconcentration in the presence of GdTex.

FIG. 8 illustrates the quantity of hydrogen peroxide generated by theaddition of LuTex to NADPH as compared to control or to a LuTex/NADPHsolution further containing catalase.

FIG. 9 illustrates the quantity of hydrogen peroxide generated by theaddition of GdTex to ascorbate as compared to control.

FIG. 10 illustrates the percent survival of MES-SA human uterine cellsin the presence of varying concentrations of ascorbate and varyingconcentrations of GdTex.

FIG. 11 illustrates the percent survival of MES-SA human uterine cellsin the presence of varying concentrations of ascorbate and varyingconcentrations of LuTex.

FIG. 12 illustrates the percent survival of EMT-6 mouse mammary sarcomacells in the presence of varying concentrations of NADPH and varyingconcentrations of LuTex.

FIG. 13 illustrates the percent survival of EMT-6 mouse mammary sarcomacells in the presence of varying concentrations of NADPH and varyingconcentrations of GdTex.

FIG. 14 illustrates the percent survival of MES-SA human uterine cellsin the presence of varying concentrations of GdTex and BSO.

FIG. 15 illustrates a proposed mechanism to explain the production ofreactive oxygen species by motexafin gadolinium and motexafin lutetium.

FIG. 16 illustrates the rate of NADPH oxidation versus time by variousmetal cation complexes of a texaphyrin.

FIGS. 17A and 17B illustrates the percent survival of MES-SA humanuterine cells with ionizing radiation and varying concentrations ofGdTex, in the absence and the presence of BSO, respectively.

FIG. 18 illustrates the clonogenic survival of MES-SA human uterinecells with ionizing radiation in the presence of GdTex and/or BSO.

FIG. 19 illustrates the percent survival of MES-SA human uterine cellstreated with BSO followed by varying concentrations of GdTex andAntimycins A.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to novel methods for treating atheroma,tumors and other neoplastic tissue as well as other conditions that areresponsive to the induction of targeted oxidative stress. However, priorto discussing this invention in further detail, the following terms willfirst be defined.

The term “texaphyrin” refers to aromatic pentadentate macrocyclic“expanded porphyrins” which are considered as being an aromaticbenzannulene containing both 18π and 22 π-electron delocalizationpathways. Such texaphryins and their synthesis are well known in theart.⁷⁻¹² Preferably, the texaphyrins encompass any and all texaphyrincompounds disclosed by Magda, et al.⁷, Young, et al.⁸, Sessler, et al.⁹⁻¹¹ and Hemmi, et al.¹²

Particularly preferred texaphyrins include those represented by formulaI:

wherein M is a divalent metal cation or a trivalent metal cation;

R¹ to R⁴ as well as R⁷ and R⁸ are independently selected from the groupconsisting of hydrogen, carboxyl, carboxylalkyl, acyl, acylamino,aminoacyl, alkyl, substituted alky (particularly hydroxyalkyl oraminoalkyl, and especially where R¹ is hydroxypropyl or aminopropyl);alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl,heteroaryl, heterocyclic, halo, hydroxyl, nitro, and a saccharide;

R⁶ and R⁹ are independently selected from the group consisting ofhydrogen, carboxyl, carboxylalkyl, acyl, acylamino, aminoacyl, alkyl,substituted alkyl other than iodoalkyl, alkenyl, substituted alkenyl,alkoxy, substituted alkoxy, aryl, heteroaryl, heterocyclic, halo otherthan iodo, hydroxyl, nitro, and a saccharide;

R⁵ and R¹⁰ to R¹² are independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy,carboxyl, carboxylalkyl, acyl and acylamino; and

the charge, Z, is an integer having a value less than or equal to 5.

The divalent or trivalent metal M is preferably selected from the groupconsisting of Ca(II), Mn(II), Co(II), Ni(II), Zn(II), Cd(II), Hg(II),Fe(II), Sm(II), UO₂(II), Mn(III), Co(III), Ni(III), Fe(III), Ho(III),Ce(III), Y(III), In(III), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III),Tb(III), Dy(III), Er(III), Tm(III), Yb(III), Lu(III), La(III), andU(III).

Particularly preferred texaphyrin compounds are represented by formulaII:

Even more preferred texaphyrin compounds are those of formula II wherein

A. M is Gd(III) and Z is +2;

B. M is Dy(III) and Z is +2;

C. M is Y(III) and Z is +2;

D. M is Lu(III) and Z is +2;

E. M is CO(II) and Z is +1;

F. M is Fe(III) and Z is +2;

G. M is Eu(III) and Z is +2;

H. M is Sm(III) and Z is +2;

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from 1 to 40 carbon atoms,more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6carbon-atoms. This term is exemplified by groups such as methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl,and the like.

The term “substituted alkyl” refers to an alkyl group as defined above,having from 1 to 5 substituents, and preferably 1 to 3 substituents,selected from the group consisting of alkoxy, substituted alkoxy,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl.

The term “alkylene” refers to a diradical of a branched or unbranchedsaturated hydrocarbon chain, preferably having from 2 to 6 carbon-atoms,more preferably 2 to 4 carbon atoms. This term is exemplified by groupssuch as ethylene (—CH₂CH₂—), the propylene isomers (e.g., —CH₂CH₂CH₂—and —CH(CH₃)CH₂—) and the like.

The term “substituted alkylene” refers to an alkylene group, as definedabove, having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl. Additionally, such substituted alkylene groupsinclude those where 2 substituents on the alkylene group are fused toform one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fusedto the alkylene group. Preferably such fused groups contain from 1 to 3fused ring structures.

The term “alkoxy” refers to the groups alkyl-O—, alkenyl-O—,cycloalkyl-O— and cycloalkenyl-O—, where alkyl, alkenyl, cycloalkyl, andcycloalkenyl are as defined herein. Preferred alkoxy groups are alkyl-O—and include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy,n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O— and substitutedcycloalkenyl-O—, where substituted alkyl, substituted alkenyl,substituted cycloalkyl, and substituted cycloalkenyl are as definedherein. A preferred class of substituted alkoxy are polyoxyalkylenegroups represented by the formula —O(R′O)_(q)R″ where R′ is an alkylenegroup or a substituted alkylene group, R″ is selected from the groupconsisting of hydrogen, alkyl or substituted alkyl and q is an integerfrom 1 to 10. Preferably, in such groups, q is from 1 to 5 and mostpreferably 3.

The term “alkenyl” refers to a monoradical of a branched or unbranchedunsaturated hydrocarbon group preferably having from 2 to 40 carbonatoms, more preferably 2 to 10 carbon atoms and even more preferably 2to 6 carbon atoms and having at least 1 and preferably from 1-6 sites ofvinyl unsaturation. Preferred alkenyl groups include ethenyl (—CH═CH₂),n-propenyl (—CH₂CH═CH₂), iso-propenyl (—C(CH₃)═CH₂), and the like.

The term “substituted alkenyl” refers to an alkenyl group as definedabove having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl.

The term “acyl” refers to the groups HC(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—,cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—, aryl-C(O)—,heteroaryl-C(O)— and heterocyclic-C(O)— where alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “acylamino” refers to the group —C(O)NRR where each R isindependently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,heterocyclic or where both R groups are joined to form a heterocyclicgroup (e.g., morpholino) wherein alkyl, substituted alkyl, aryl,heteroaryl and heterocyclic are as defined herein.

The term “aminoacyl” refers to the group —NRC(O)R where each R isindependently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, orheterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

The term “aminoacyloxy” refers to the group —NRC(O)OR where each R isindependently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, orheterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclic-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclic are as defined herein.

The term “aryl” refers to an unsaturated aromatic carbocyclic group offrom 6 to 20 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed (fused) rings (e.g., naphthyl or anthryl). Preferredaryls include phenyl, naphthyl and the like.

Unless otherwise constrained by the definition for the aryl substituent,such aryl groups can optionally be substituted with from 1 to 5substituents, preferably 1 to 3 substituents, selected from the groupconsisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl,cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl,amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy,azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino,thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro,trihalomethyl, and thioalkoxy.

The term “aryloxy” refers to the group aryl-O— wherein the aryl group isas defined above including optionally substituted aryl groups as alsodefined above.

The term “amino” refers to the group —NH₂.

The term “substituted amino refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, aryl,heteroaryl and heterocyclic provided that both R's are not hydrogen.

The term “carboxyalkyl” refers to the groups “—C(O)O-alkyl”,“—C(O)O-substituted alkyl”, “—C(O)O-cycloalkyl”, “—C(O)O-substitutedcycloalkyl”, “—C(O)O-alkenyl”, and “—C(O)O-substituted alkenyl”, wherealkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,and substituted alkenyl, are as defined herein.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 20carbon atoms having a single cyclic ring or multiple condensed rings.Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, and preferably 1 to 3 substituents, selectedfrom the group consisting of alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 20carbon atoms having a single cyclic ring and at least one point ofinternal unsaturation. Examples of suitable cycloalkenyl groups include,for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and thelike.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, and preferably 1 to 3 substituents, selectedfrom the group consisting of alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

The term “heteroaryl” refers to an aromatic group of from 1 to 15 carbonatoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfurwithin at least one ring (if there is more than one ring).

Unless otherwise constrained by the definition for the heteroarylsubstituent, such heteroaryl groups can be optionally substituted with 1to 5 substituents, preferably 1 to 3 substituents, selected from thegroup consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy,alkenyl, cycloalkyl, cycloalkenyl, substituted alkyl, substitutedalkoxy, substituted alkenyl, substituted cycloalkyl, substitutedcycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl,aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro,heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy,oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl and trihalomethyl. Preferred aryl substituents includealkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy. Suchheteroaryl groups can have a single ring (e.g., pyridyl or furyl) ormultiple condensed rings (e.g., indolizinyl or benzothienyl). Preferredheteroaryls include pyridyl, pyrrolyl and furyl.

The term “heteroaryloxy” refers to the group heteroaryl-O—.

The term “heterocycle” or “heterocyclic” refers to a monoradicalsaturated unsaturated group having a single ring or multiple condensedrings, from 1 to 40 carbon atoms and from 1 to 10 hetero atoms,preferably 1 to 4 heteroatoms, selected from nitrogen, sulfur,phosphorus, and/or oxygen within the ring.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, and preferably 1 to 3 substituents, selected from the groupconsisting of alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Suchheterocyclic groups can have a single ring or multiple condensed rings.Preferred heterocyclics include morpholino, piperidinyl, and the like.

Examples of nitrogen heterocycles and heteroaryls include, but are notlimited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,piperidine, piperazine, indoline, morpholino, piperidinyl,tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containingheterocycles.

The term “heterocyclooxy” refers to the group heterocyclic-O—.

The term “thioheterocyclooxy” refers to the group heterocyclic-S—.

The term “oxyacylamino” refers to the group —OC(O)NRR where each R isindependently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, orheterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

The term “thiol” refers to the group —SH.

The term “thioalkoxy” refers to the group —S-alkyl.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined above including optionally substituted aryl groupsalso defined above.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined above including optionallysubstituted aryl groups as also defined above.

The term “saccharide” refers to oxidized, reduced or substitutedsaccharides hexoses such as D-glucose, D-mannose, D-xylose, D-galactose,D-glucuronic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine,sialyic acid, iduronic acid, L-fucose, and the like; pentoses such asD-ribose or D-arabinose; ketoses such as D-ribulose or D-fructose;disaccharides such as sucrose, lactose, or maltose; derivatives such asacetals, amines, acylated, sulfated and phosphorylated sugars;oligosaccharides having from 2 to 10 saccharide units. For the purposesof this definition, these saccharides are referenced using conventionalthree letter nomenclature and the saccharides can be either in theiropen or preferably in their pyranose form.

As to any of the above groups that contain one or more substituents, itis understood, of course, that such groups do not contain anysubstitution or substitution patterns which are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers and mixtures thereofarising from the substitution of these compounds.

The term “treatment” or “treating” means any treatment of a disease in amammal, including:

(i) preventing the disease, that is, causing the clinical symptoms ofthe disease not to develop;

(ii) inhibiting the disease, that is, arresting the development ofclinical symptoms; and/or

(iii) relieving the disease, that is, causing the regression of clinicalsymptoms.

The term “effective amount” means a dosage sufficient to providetreatment for the disease state being treated. This will vary dependingon the patient, the disease and the treatment being effected.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions well known in the artand include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate,maleate, oxalate and the like.

The term “cellular metabolite” or “reducing metabolite” refers to acompound found naturally within a living cell. The cellular metabolitesemployed to generate reactive oxygen species by the methods disclosedherein having a standard biochemical reduction potential more negativethan the standard biochemical reduction potential of oxygen/hydrogenperoxide. Such metabolites include, by way of example only, NAD(P)H(i.e., NADPH and/or NADH), FADH₂, ascorbate, reduced glutathione,dihydrolipoic acid and the like.

The term “standard biochemical reduction potential” refers to thereduction potential of a metabolite measured at pH 7 and 25° C. in anaqueous solution. At these conditions, oxygen and hydrogen peroxide havea reduction potential of approximately 0.273 mV.¹⁶

The term “thiol-depleting compound” refers to a compound which uponadministration to a host or to a cell, results in a global lowering ofthe concentration of available reduced thiol (e.g., glutathione).Examples of thiol-depleting compounds include buthionine sulfoximine(“BSO”, a known inhibitor of glutathione synthesis), diethyl maleate (athiol reactive compound) dimethyl fumarate, N-ethyl maleimide, diamide(diazene dicarboxylic acid bis-(N,N′-dimethylamide)) and the like.

The term “ionizing radiation” refers to radiation conventionallyemployed in the treatment of tumors which radiation, either as a largesingle dosage or as repeated smaller dosages, will initiate ionizationof water thereby forming reactive oxygen species. Ionizing radiationincludes, by way of example, x-rays, electron beams, γ-rays, and thelike.

The term “porphyrin derivative” refers to those molecules which containas part of their chemical structure a polypyrrole macrocycle.

The-term “DNA alkylators” refer to-well known alkylating agents whichalkylate DNA thereby interfering with cellular processes and leading tocell death. Suitable alkylating agents include nitrogen mustards (e.g.,mechlorethamine, cyclophosphamide, ifosfamide, mephalan, chlorambuciland estramustine), etheleneimines and methylmelamines (e.g.,hexamethylmelamine and thiotepa), alkyl sulfonates (e.g., busulfan),nitroureas (e.g., carmustine, lomusine, semustine, and streptozocin),and triazines (e.g., dacarbazine, procarbazine, and aziridine).

The term “topoisomerase” refers to enzymes that control and modify thetopological states of DNA by catalyzing the concerted breaking andrejoining of DNA strands. (see, for example, D'Arpa et al., Biochim.Biophys. Acta, 989, 163 (1989). At least two distinct topoisomerases areknown in the art and are designated as topoisomerase I and II.

The term “topoisomerase inhibitors” refers to compounds which in vivoinhibit one or more of the topoisomerase enzymes. Topoisomerase IIinhibitors are well known in the art and include, by way of example,etoposide (VP-16), teniposide (VM-26), mitoxantrone, m-AMSA,adriamycin(doxorubicin), ellipticine and daunomycin. Topoisomerase Iinhibitors are also well known in the art and include, for example,camptothecin and Hoechst 33342. See, for instance, Allan Y. Chen, etal., “A New Mammalian DNA Topoisomerase I Poison Hoechst 33342:Cytoxicity and Drug Resistance in Human Cell Cultures”, Cancer Research,53:1332-1337 (1993). Still other topoisomerase I and II inhibitors aredisclosed in U.S. Pat. No. 5,807,874; by Darshan Makhey, et al.,“Coralyne and Related Compounds as Mammalian Topoisomerase I andTopoisomerase II Poisons”, Bioorg. & Med. Chem. Lett., 4:781-791, (1996)and Darshan Makhey, et al., “Protoberberine Alkaloids and RelatedCompounds as Dual Inhibitors of Mammalian Topoisomerase I and II”, Med.Chem. Res., 5:1-12 (1994). All of these references are incorporatedherein by reference in their entirety.

The term “redox cycling agents” refers to compounds which may exist intwo or more oxidation states and are able to lower the activationbarrier for electron transfer between two compounds. Examples ofsuitable redox cycling agents include, for instance, alloxan, phenazinemethosulfate, menadione, copper/putrescine/pyridine, methylene blue,paraquat, doxorubicin, bleomycin, and ruthenium (II)tris-(1,10-phenanthroline-5,6-dione).

The term “metabolic inhibitors” or “antimetabolites” refer to materialswhich interfere with the availability of one or more cellularmetabolites such as ascorbate, NAD(P)H, etc. Such metabolic inhibitorsare well known in the art and include, by way of example, folic acidanalogs (e.g., methotrexate and trimetrexate), pyrimidine analogs (e.g.,fluorouracil, floxuridine, cytarabine and azacitidine), and purineanalogs and related inhibitors (e.g., mercaptopurine, thioguanine,pentostatin and fludarabine).

The term “mitochondrial inhibitors” refers to compounds that interferewith mitochondrial metabolism including, for example, oligomycin (aspecific inhibitor of motochondrial ATP-ase, i.e., complex 5) andantimycins A (a complex 3 inhibitor that blocks the conversion ofubiquinol to ubiquinone).

Methods

Compounds that display affinity for electrons can potentiate thebiological effect of ionizing radiation and, accordingly, may have useas radiation sensitizers. The determination of which compoundsdisplaying electron affinity may possess radiation sensitizationproperties was heretofore not possible absent in vivo trial and errortesting. Indeed, motexafin gadolinium [PCI-0120, Xcytrin™, GdTex,Formula II where M is Gd(III)] is reported to enhance the efficacy ofradiation in animal tumor models and is currently in Phase III clinicaldevelopment as an adjuvant to radiation therapy.^(11,13) However, therelated lutetium(III) congener (PCI-0123, Lutrin™, LuTex) in the sameanimal model did not possess similar radiation sensitization properties.These results have led us to explore the chemical mechanisms for theobserved biological activity of GdTex.

Biochemically, two cellular forms of rapidly accessible energy aremaintained for metabolic use: high energy phosphoryl linkages [e.g.,phosphocreatine, ATP) and electron carriers, i.e., the cofactorsNAD(P)H]. Initially, texaphyrins were evaluated for their ability toalter cellular energy charge by catalyzing the hydrolysis of high energyphosphoryl linkages. The results of this evaluation suggested someapparent effect; however, the observed rate accelerations seemedincompatible with biological importance.

Co-incubation of NADH or NADPH with texaphyrin derivatives, however,rapidly converted these cofactors to their oxidized forms, at catalyticconcentrations of complex. Examination of texaphyrins containingdifferent lanthanide ions showed that the observed oxidation ratesmeasured by the amount of NADP+ formed correlated with Lewis acidity,whereas the transition metal derivatives displayed a lack of reactivitywhich is consistent with the fact that the redox potentials oftexaphyrin complexes of divalent cations are about 200 mV more negativethan those of the trivalent cations. It is also consistent with thelower attraction of oxygen anion ligands for transition metal cationsthan for lanthanide cations. The results of this examination aredepicted in FIGS. 1-6.

Without being limited to any theory, these findings suggested that thereaction proceeded via initial complexation of cofactor phosphoricanhydride with the metal center, followed by slow two-electron (orhydride) transfer to the texaphyrin macrocycle. Indeed, the reactiondisplayed saturation kinetics.

The above studies were conducted under aerobic conditions. Replacementof oxygen with inert atmosphere led to rapid texaphyrin complexdegradation, as evidenced by bleaching of the characteristic texaphyrinuv-vis absorbance spectrum (data not shown). Again, without beinglimited to any theory, this led to the supposition that oxygen mightserve as the ultimate electron acceptor under aerobic conditions.

As illustrated in Examples 1-4 below, this supposition was assessed bymeasuring the formation of oxidized species or hydrogen peroxide uponincubation of the cellular metabolite NADPH with LuTex (Examples 1 and3) or the cellular metabolite ascorbate with GdTex (Examples 2 and 4).As appropriate, the addition of catalase to the reaction mixtureconfirmed that the assay signal generated therein was due to hydrogenperoxide. In each case, the cellular metabolites, NADPH and ascorbate,have a standard biochemical reduction potential more negative than thestandard biochemical reduction of oxygen/hydrogen peroxide.

These results evidenced that a compound's potential to exhibit radiationsensitizing activity correlates to its ability to form one or morereactive oxygen species from cellular metabolites which have a standardbiochemical reduction potential more negative than the standardbiochemical reduction of oxygen/hydrogen peroxide. In turn, thisprovides a facile method for testing a compounds radiation sensizitingcapacity by introducing the to be tested compound into an aqueoussolution comprising a cellular metabolite having a standard biochemicalreduction potential more negative than the standard biochemicalreduction potential of oxygen/hydrogen peroxide; monitoring the solutionfor the occurrence of a reaction that produces one or more reactiveoxygen species; and determining whether the compound has probableradiation sensitization activity, wherein this activity correlates tothe occurrence and extent of a reaction that produces reactive oxygenspecies.

As is apparent, this assay can monitor any of a number of differentcomponents to assess the extent of the reaction. For example, thedepletion of the cellular metabolite can be assayed; the production ofan oxidized form of cellular metabolite can be assayed; the depletion ofoxygen from the reaction solution can be assayed; or the appearance ofhydrogen peroxide can be assayed. Each of these assays can, in turn, beused to determine the extent of reaction. As above, the cellularmetabolite employed is one that has a standard biochemical reductionpotential more negative than the standard biochemical reduction ofoxygen/hydrogen peroxide. Such metabolites include, by way of exampleonly, NADPH, NADH, FADH, ascorbate and reduced glutathione.

In a particularly preferred embodiment, compounds determined to haveradiation sensitization activity by virtue of their ability to generateone or more reactive oxygen species in vivo are useful as adjuncts totreating mammalian tumors by ionizing radiation. In this embodiment, thecompound possessing radiation sensitization activity is administered tothe tumor in sufficient quantities to therapeutically enhance the effectof ionizing radiation on tumor cell death.

For example, the proliferation of human ovarian cancer cell line MES-SA⁷can be used to assess the degree of ascorbate and cofactor oxidationunder cell culture conditions. RPMI 1640, which contains no ascorbate,was used as the medium in these experiments. Specifically, asillustrated in Examples 3 and 4, coincubation of ascorbate and GdTex orLuTex resulted in decreased cell proliferation, as measured bytetrazolium salt (MTT) reduction,¹⁴ due to hydrogen peroxide formation.This occurred at concentrations of ascorbate, which parallel thedissociation constant values found. Similar results were obtained usingNADPH as substrate.

Particularly useful radiation sensitizers are compounds thatpreferentially localize in the tumors. For example, it is well knownthat texaphyrin and porphyrin compounds will preferentially localize inmammalian tumors and have potential radiation sensitization activity.Similarly, other compounds determined to have radiation sensitizationactivity may also preferentially localize in mammalian tumors or suchcompounds can be derivatized to impart preferential localization inmammalian tumors. For example, such compounds can be derivatized byconventional synthetic chemical techniques to append to a molecule whichis known to localize in mammalian tumors. Such molecules includemonoclonal antibodies directed to tumor antigens, texaphyrins,porphyrins, peptides such as disclosed by Urzgiris, et al., U.S. Pat.No. 5,762,909 which is incorporated herein by reference in its entirety,etc. Specific techniques for coupling such compounds are disclosed inU.S. patent application Ser. No. 09/431,298, filed Oct. 29, 1999 andentitled “Compounds for Treating Atheroma, Tumors and other NeoplasticTissue ” which application is incorporated herein by reference in itsentirety.

One preferred compound for use as a radiation sensitizer are porphyrinderivatives and, in particular, iron(III) porphyrin. Such derivativesare known to accumulate in tumor tissue and iron(III) porphyrin has beendisclosed as generating hydrogen peroxide from ascorbate and oxygen.¹⁵

Alternatively, the generation of one or more reactive oxygen species bythe radiation sensitizers of the present invention can be used by itself(or in conjunction with the administration of a reducing metabolite) totherapeutically treat a tumor or atheroma. When used in conjunction withthe administration of such reducing metabolites, the radiationsensitizers encompassed by the present invention exclude the cobalt andiron complexes of phthalocyanine and napthalocyanine. In one aspect ofthe invention, this can be particularly useful when the patient has beenexposed to the maximum amount of ionizing radiation which can betolerated by the patient.

Further, the role of glutathione and ascorbate towards lesions inducedby ionizing radiation has been well studied.¹¹ It is generally acceptedthat a competition exists between these species and molecular oxygen forDNA lesions, such that a protective role for glutathione (and, at lowlevels of glutathione, ascorbate) can be demonstrated under anoxicconditions. As is known in the art, glutathione imparts a protectiverole by virtue of a redox reaction wherein the free thiol group ofglutathione is oxidized to form a disulfide linkage with a secondoxidized glutathione molecule. Accordingly, in order to limit theprotection afforded by gluathione or other thiol containingantioxidants, this invention contemplates co-administraton of aneffective amount of a thiol-depleting agent to the tumor cell or to apatient suffering from cancer in order to reduce the amount of thiolcontaining antioxidants contained therein. Preferably thethiol-depleting agent is buthionine sulfoximine.

Alternatively, known chemotherapeutic agents or other agents which altermetabolic pathways can be co-administered with the radiation sensitizingcompound. Such agents include, by way of example, DNA alkylators,topoisomerase inhibitors, redox cycling agents, metabolic inhibitors andmitochondrial inhibitors.

The metabolic balance of a cell entails numerous synchronous reactions.FIG. 15 illustrates pathways entailing the cellular metabolitesascorbate and NADPH, both of which have a standard biochemical reductionpotential more negative than the standard biochemical reductionpotential of oxygen/hydrogen peroxide. Agents capable of catalyzing theproduction of one or more reactive oxygen species from either or both ofthese cellular metabolites can drive the cell toward a state ofoxidative stress. Also illustrated are certain of the effects ofionizing radiation, e.g., leading to the generation of hydroxyl radicalsand additional hydrogen peroxide. Employing the methods of the presentinvention, it has been determined that motexafin gadolinium catalyzesthe production of hydrogen peroxide from ascorbate to a much greaterextent than does motexafin lutetium, whereas the converse is true withrespect to NADPH. This suggests the co-administration of motexafingadolinium and motexafin lutetium to drive both of these pathways as ameans of increasing oxidative stress to increase a cell's sensitivity toradiation.

The preferred pharmaceutical compositions and methods of treatment ofthe present invention include the following:

-   -   Co-administration of a texaphyrin and a thiol-depleting agent, a        reducing metabolite or source thereof, or a mitochondrial        inhibitor, with or without ionizing radiation.    -   Co-administration of a texaphyrin and a thiol-depleting agent,        with or without ionizing radiation; particularly where the        thiol-depleting agent is BSO and most preferably with ionizing        radiation. Especially preferred are the co-administration of a        thiol-depleting agent with GdTex, CoTex, the mu-oxo dimer of        iron texaphyrin (“Fe(Tex)₂O”), EuTex, SmTex, di-amino GdTex and        di-amino LuTex, again, more preferably where the thiol-depleting        agent is BSO and most preferably with ionizing radiation. The        co-adminstration of GdTex and BSO, followed by administration of        ionizing radiation, is most preferred.    -   Co-administration of a texaphyrin and a reducing metabolite or        source thereof, with or without ionizing radiation, particularly        where the texaphyrin is co-administered with ascorbate.        Especially preferred are the co-administration of a reducing        metabolite with GdTex, CoTex, the mu-oxo dimer of iron        texaphyrin (“Fe(Tex)₂O”), EuTex, SmTex, di-amino GdTex and        di-amino LuTex, again, more preferably where the reducing        metabolite is ascorbate and most preferably with ionizing        radiation. The co-adminstration of GdTex and ascorbate, followed        by administration of ionizing radiation, is most preferred.    -   Co-administration a texaphyrin (particularly GdTex), BSO and        ascorbate, with or without ionizing radiation, and most        preferably followed by ionizing radiation.    -   Co-administration of a non-texaphyrin compound that catalyzes        the production of one or more reactive oxygen species from a        cellular metabolite having a standard biochemical reduction        potential more negative than the standard biochemical reduction        of oxygen/hydrogen peroxide and ionizing radiation.    -   Co-administration of a DNA alkylator, thiol-depleting agent,        topoisomerase inhibitor, redox cycling agent, metabolic        inhibitor and/or mitochondrial inhibitor and a non-texaphyrin        compound that catalyzes the production of one or more reactive        oxygen species from a cellular metabolite having a standard        biochemical reduction potential more negative than the standard        biochemical reduction of oxygen/hydrogen peroxide, with or        without ionizing radiation (excluding ascorbic acid with cobalt        or iron phthalocyanines and naphthalocyanines without ionizing        radiation).    -   Co-administration of a redox cycling agent and a non-texaphyrin        compound that catalyzes the production of one or more reactive        oxygen species from a cellular metabolite having a standard        biochemical reduction potential more negative than the standard        biochemical reduction of oxygen/hydrogen peroxide, with or        without ionizing radiation, particularly where the redox cycling        agent is doxorubicin or bleomycin, and most particularly with        ionizing radiation. Especially preferred is the non-texaphyrin        compound methylene blue, co-administered with bleomycin or        doxorubicin, and without ionizing radiation.

Excluded from the pharmaceutical compositions and methods of treatmentof the present invention are those combinations of texaphyrins (such asmotexafin gadolinium), chemotherapeutic agents (such as doxorubicin)and/or co-therapeutic agents (such as ionizing, photodynamic andsonodynamic energy sources) previously disclosed, for example, in U.S.Pat. No. 5,776,925 and in WO00/01414. However, to the extent thatpharmacologically active mediators of oxidative stress have notpreviously been disclosed for administration in combination with atexaphyrin, such as thiol-depleting agents (especially BSO),mitochondrial inhibitors (such as antimycins A) and certain redoxcycling agents (e.g., excluding doxorubicin) are intended to be withinthe scope of the invention. Similarly excluded is the co-administrationof ascorbic acid with cobalt or iron phthalocyanines andnaphthalocyanines (e.g., as disclosed in U.S. Pat. No. 6,004,953), but,not when combined with the administration of ionizing radiation.

Utility

Methods for determining compounds which are useful as radiationsensitizers provide a facile means to assess the potential of suchcompound for use as adjuncts in treating tumors with ionizing radiation.In addition, the fact that such compounds produce one or more reactiveoxygen species in vivo dictates that these compounds will be useful intheir own right in killing cells such as tumor cells even in the absenceof ionizing radiation.

When employed with ionizing radiation, the amount of compoundadministered to the patient will vary depending upon what is beingadministered, the purpose of the administration, the state of thepatient, the manner of administration, and the like. In particular, asufficient amount of the compound is administered to the cell or to thepatient to therapeutically enhance the effect of ionizing radiation ontumor cell death. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on the judgment of the attending clinician depending upon factorssuch as the degree or severity of the cancer in the patient, the age,weight and general condition of the patient, and the like. Preferably,radiation sensitizing compounds used in conjunction with ionizingradiation are administered at dosages ranging from about 0.1 to about100 mg/kg/day. Preferably, the active agent is administeredapproximately 2-5 hours prior to exposure to ionizing radiation.

When employed in the absence of ionizing radiation, the amount ofcompound administered to the patient will again vary depending upon whatis being administered, the purpose of the administration, the state ofthe patient, the manner of administration, and the like. In particular,a sufficient amount of the compound is administered to the cell or tothe patient so as to generate reactive oxygen species in quantitieseffective to initiate tumor cell death. An amount adequate to accomplishthis is defined as “therapeutically effective dose.”Amounts effectivefor this use will depend on the judgment of the attending cliniciandepending upon factors such as the degree or severity of the cancer inthe patient, the age, weight and general condition of the patient, andthe like. Preferably, when so employed, the compound is administered atdosages ranging from about 0.1 to about 100 mg/kg/day.

Similarly, drugs co-administered to the patient such as DNA alkylators,topoisomerase inhibitors, redox cycling agents, thiol-depleting agentsand metabolic inhibitors are also employed in sufficient quantities fortheir intended purpose. These amounts are well documented in the art.

As noted above, the compounds administered to a patient are in the formof pharmaceutical compositions described herein. These compositions maybe sterilized by conventional sterilization techniques, or may besterile filtered. When aqueous solutions are employed, these may bepackaged for use as is, or lyophilized, the lyophilized preparationbeing combined with a sterile aqueous carrier prior to administration.The pH of the compound preparations typically will be between 3 and 11,more preferably from 5-9 and most preferably from 7 and 8. It will beunderstood that use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of pharmaceutical salts.

Pharmaceutical Formulations

When employed as pharmaceuticals, compounds described herein are usuallyadministered in the form of pharmaceutical compositions. These compoundscan be administered by a variety of routes including oral, intravenous,intramuscular, and the like. These compounds are effective as bothinjectable and oral compositions. Such compositions are prepared in amanner well known in the pharmaceutical art and comprise at least oneactive compound.

These pharmaceutical compositions contain, as the active ingredient, oneor more of the compounds described herein associated withpharmaceutically acceptable carriers. In making these compositions, theactive ingredient is usually mixed with an excipient. When the excipientserves as a diluent, it can be a solid, semi-solid, or liquid material,which acts as a vehicle, carrier or medium for the active ingredient.Thus, the compositions can be in the form of tablets, pills, elixirs,suspensions, emulsions, solutions, syrups, and the like containing, forexample, up to 10% by weight of the active compound, soft and hardgelatin capsules, sterile injectable solutions, and sterile packagedpowders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 5 to about 100 mg, more usually about 10 toabout 30 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient. Preferably, the active ingredient is employed at no more thanabout 20 weight percent of the pharmaceutical composition, morepreferably no more than about 15 weight percent, with the balance beingpharmaceutically inert carrier(s).

An active compound is typically effective over a wide dosage range andis generally administered in a pharmaceutically effective amount. It,will be understood, however, that the amount of the active ingredientactually administered will be determined by a physician, in the light ofthe relevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

By way of example, the radiation sensitizer motexafin gadolinium isadministered in a solution containing 2 mM optionally in 5% mannitolUSP/water (sterile and non-pyrogenic solution). Dosages of 0.1 mg/kg upto as high as about 23.0 mg/kg have been delivered, preferably about 3.0to about 15.0 mg/kg (for volume of about 90 to 450 mL) may be employed,optionally with pre-medication using anti-emetics above about 6.0 mg/kg.The texaphyrin is administered via intravenous injection over about a 5to 10 minute period, followed by a waiting period of about 2 to 5 hoursto facilitate intracellular uptake and clearance from the plasma andextracellular matrix prior to the administration of radiation.

When employing radiation therapy, a palliative course of 30 Gy in ten(10) fractions of radiation are typically administered over consecutivedays excluding weekends and holidays. In the treatment of brainmetastases, whole brain megavolt radiation therapy is delivered with 60Co teletherapy or a ≧4 MV linear accelerator with isocenter distances ofat least 80 cm, using isocentric techniques, opposed lateral fields andexclusion of the eyes. A minimum dose rate at the midplane in the brainon the central axis is about 0.5 Gy/minute.

Radiation sensitizers may be administered before, or at the same timeas, or after administration of the ionizing radiation, preferablybefore. The radiation sensitizer may be administered as a single dose,as an infusion, or it may be administered as two or more doses separatedby an interval of time. Where the radiation sensitizer is administeredas two or more doses, the time interval between administrations may befrom about one minute to a number of days, preferably from about 5 minto about 1 day, more preferably about 4 to 5 hr. The dosing protocol maybe repeated, from one to ten or more times, for example. Dose levels forradiation sensitization using motexafin gadolinium may range from about0.05 μmol/kg to about 20 μmol/kg administered in single or multipledoses (e.g. before each fraction of radiation). A lower dosage range ispresently preferred for intra-arterial injection or for impregnatedstents. In the case of texaphyrins incorporating or conjugated to aradioisotope, the additional administration of radiation as aco-therapeutic agent is optional.

Administering a radiation sensitizer to a mammalian host bearingatheroma cells may be prior to, concurrent with, or following vascularintervention, and the intervention is followed by radiation. Theadministration may begin prior to, such as about 24-48 hours prior to,or at a time roughly accompanying vascular intervention, for example.Multiple or single treatments prior to, at the time of, or subsequent tothe procedure may be used. “Roughly accompanying the vascularintervention” refers to a time period within the ambit of the effects ofthe vascular intervention. Typically, an initial dose of the sensitizerand radiation will be within 1-24 hours of the vascular intervention,preferably within about 5-24 hours thereafter. Follow-up dosages may bemade at weekly, biweekly, or monthly intervals. Design of particularprotocols depends on the individual subject, the condition of thesubject, the design of dosage levels, and the judgment of the attendingpractitioner. In the methods of the invention involving theadministration of a compound that catalyzes the production of one ormore reactive oxygen species from a cellular metabolite having astandard biochemical reduction potential more negative than the standardbiochemical reduction of oxygen/hydrogen peroxide, a source or precursorof such cellular metabolite is co-administered either before,contemporaneously with the catalytic agent or subsequent to itsadministration; either or both agents may be administered systemicallyor locally (e.g., by intra-arterial injection).

The following formulation examples illustrate representativepharmaceutical compositions of the present invention.

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

Formulation Example 2

A tablet formula is prepared using the ingredients below:

Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

Formulation Example 3

Tablets, each containing 30 mg of active ingredient, are prepared asfollows:

Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mg Starch 45.0 mgMicrocrystalline cellulose 35.0 mg Polyvinylpyrrolidone 4.0 mg (as 10%solution in sterile water) Sodium carboxymethyl starch 4.5 mg Magnesiumstearate 0.5 mg Talc 1.0 mg Total 120 mg

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50 to 60 C and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

Formulation Example 4

Capsules, each containing 40 mg of medicament are made as follows:

Quantity Ingredient (mg/capsule) Active Ingredient  40.0 mg Starch 109.0mg Magnesium stearate  1.0 mg Total 150.0 mg

The active-ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 150 mg quantities.

Formulation Example 5

Suspensions, each containing 50 mg of medicament per 5.0 mL dose aremade as follows:

Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodiumcarboxymethyl cellulose (11%) 50.0 mg Microcrystalline cellulose (89%)Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v. Purifiedwater to 5.0 mL

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

Formulation Example 6

Capsules are made, as follows:

Quantity Ingredient (mg/capsule) Active Ingredient  15.0 mg Starch 407.0mg Magnesium stearate  3.0 mg Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 425.0 mg quantities.

Formulation Example 7

An injectable preparation buffered to a pH of 7.4 is prepared having thefollowing composition:

Ingredients Amount Active Ingredient 0.2 g Sodium Phosphate BufferSolution (0.8 M) 10.0 ml DMSO 1.0 ml WFI q.s. to 100 ml

Formulation Example 8

An injectable formulation is prepared having the following composition:

Ingredients Amount (w/v %) Motexafin gadolinium 0.23% Motexafin lutetium0.20% Mannitol (USP) 5.0% Acetic Acid (5%) adjust to pH 5.4 Sterile WFI(USP) q.s. to 100%The formulation is filled into a glass vials, which are then purged withnitrogen to exclude oxygen from the head space and then sealed.

It may be desirable or necessary to introduce the pharmaceuticalcomposition to the brain, either directly or indirectly. Directtechniques usually involve placement of a drug delivery catheter intothe host's ventricular system to bypass the blood-brain barrier. Onesuch implantable delivery system used for the transport of biologicalfactors to specific anatomical regions of the body is described in U.S.Pat. No. 5,011,472 which is herein incorporated by reference.

Indirect techniques, which are generally preferred, usually involveformulating the compositions to provide for drug latentiation by theconversion of hydrophilic drugs into lipid-soluble drugs. Latentiationis generally achieved through blocking of the hydroxy, carbonyl,sulfate, and primary amine groups present on the drug to render the drugmore lipid soluble and amenable to transportation across the blood-brainbarrier. Alternatively, the delivery of hydrophilic drugs may beenhanced by intra-arterial infusion of hypertonic solutions which cantransiently open the blood-brain barrier.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985).

The following examples are offered to illustrate this invention and arenot to be construed in any way as limiting the scope of this invention.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning.

-   -   BSO=buthionine sulfoximine    -   CdTex=compound of formula II where M is Cd²⁺    -   CoTex=compound of formula II where M is Co³⁺    -   Di-amino GdTex=compound of formula II where M is Gd³⁺ except        that both R¹ (as shown in formula I) is aminopropyl    -   Di-amino LuTex=compound of formula II where M is Lu³⁺ except        that both R¹ (as shown in formula I) is aminopropyl    -   DyTex=compound of formula II where M is Dy³⁺    -   EuTex=compound of formula II where M is Eu³⁺    -   Fe(Tex)₂O=mu-oxo dimer of two compounds of formula II where M is        Fe²⁺    -   GdTex=motexafin godolinium (formula II where M is Gd³⁺)    -   HEPES=hydroxyethylpiperizine ethane sulfonic acid    -   HPLC=high performance liquid chromatography    -   LuTex=motexafin lutetium (formula II where M is Lu³⁺)    -   mg=milligram    -   mL=milliliter    -   mm=millimeter    -   mM=millimolar    -   MnTex=compound of formula II where M is Mn²⁺    -   mmol=millimols    -   nm=nanometer    -   psi=pounds per square inch    -   RPMI 1640=a commercially available culture medium    -   SmTex=compound of formula II where M is Sm³⁺    -   ′YTex=compound of formula II where M is Y³⁺    -   μL=microliter    -   μM=micromolar

Example 1 Oxidation of NADPH under Approximate Physiologic Conditions

This example measures radiation sensitization potential as a function ofoxidation of the cellular metabolite NADPH.

1A. Materials and Method.

The following stock solutions were prepared:

-   -   Stock NADPH (Sigma N 7505) (prepared fresh daily): 12.2 mg/(10        mL. 833.4 mg/mmol)=1.46 mM    -   Stock GdTex: 5.76 mg/(10.0 mL water 1148 mg/mmol)=501.7 μM    -   Stock 4× Buffer: 200 mM HEPES, pH 7.5; 400 mM NaCl; 0.4 mM EDTA    -   Stock LuTex: 5.02 mg/(10.0 mL water×1166 mg/mmol)=431 μM        These were combined to form the reaction mixtures analyzed by        HPLC, for example:    -   250 μL 4× Buffer, 250 μL NADPH Stock, 427 μL Water, 72.7 μL        GdTex Stock    -   250 μL 4× Buffer, 250 μL NADPH Stock, 457.7 μL Water, 42.3 μL        LuTex Stock        The HPLC Method employed was as follows:

-   1. HPLC Operating Parameters:    -   System—HPLC system capable of delivering gradient mobile phase    -   Detector wavelength—260 nm    -   Injection Volume—20 μL    -   Pressure—ca: 2000 psi    -   Column Temperature—40° C.    -   Column—Nucleogel DEAE 60-7 125×4 mm

-   2. Mobile Phase:    -   Solution A—0.75 M KH₂PO₄, pH 3.8    -   Solution B—0.016 M KH₂PO₄, pH 3.8

Time(min.) Flow (mL/min) A(%) B(%) inital 1.0 5 95 5 1.0 5 95 30 1.0 8020 31 1.5 5 95 39 1.0 5 95 40 1.0 5 95

A reaction mixture was prepared containing NADPH, water and sufficientbuffer to maintain the solution at pH 7.5. Texaphyrin, 0.1 molarequivalent to the NADPH, was added to this reaction mixture, vortexedbriefly, and then the solution placed in the HPLC sampler for immediateinjection. Further injections were made at appropriate time points,whereupon the integrated peak areas of both NADPH and NADP⁺ weremeasured and used to calculate the extent of reaction at a given timepoint.

Provided that the amount of texaphyrin was sufficiently low relative tosubstrate, the percentage of NADP⁺ generated by the reaction plottedagainst time initially gave a straight line. (For LuTex, it wasnecessary to drop the catalyst concentration to ca. 0.05 equivalent.)The slope of initial time points (eg., plotting as mM concentration vstime in hours) provided a rate which was used as a point in a saturation(Michaelis-Menton) plot. This process was repeated at variousconcentrations of NADPH. Data (in triplicate) was fitted to theMichaelis-Menton equation (initial reaction velocity/catalystconcentration)=(k_(cat)×substrate concentration)/(K_(M)+substrateconcentration), where substrate is NADPH, catalyst is texaphyrincomplex, k_(cat) is the first-order rate constant for the catalyst, andK_(M) is the dissociation (or Michaelis) constant for thecatalyst/substrate complex. For GdTex, k_(cat)=0.049±0.003 min⁻¹ andK_(M)=1.69±0.17 mM. For LuTex, k_(cat)=0.119±0.011 min⁻¹ andK_(M)=0.56±0.20 mM.

Results. The results of this analysis are depicted in FIGS. 1-6. FIGS.1-3 illustrate triplicate runs for LuTex which runs are recited as LuTexA, LuTex B and LuTex C. FIGS. 4-6 illustrate triplicate runs for GdTexwhich runs are recited as GdTex A, GdTex B and GdTex C

These results evidence that in the presence of texaphyrins, NADPH wasoxidized to NADP⁺, and further that the test compounds have probableradiation sensitization activity.

1B. By following the procedure of Example 1A using a singleconcentration of NADPH and employing 0.1 equivalent of test compound,the results illustrated in FIG. 16 were obtained. These results evidencethat in the presence of the tested texaphyrins, NAPDH was oxidized toNADP⁺, and further that the test compounds (with the exception ofMn²⁺Tex) have probable radiation sensitization activity where NADPH isthe reducing metabolite.

Example 2 Oxidation of Ascorbate under Approximate PhysiologicConditions

This example measures radiation sensitization potential as a functionthe oxidation of ascorbate, employing an assay modified from Buettner,et al., Radiation Research 145:532-541 (1996).

2A. Materials and Method.

The following stock solutions were prepared:

-   -   Stock ascorbic acid (Sigma A 5960) (prepared fresh): 8.82 mg/(25        mL 176.1 mg/mmol)=2.00 mM    -   Stock GdTex: 5.72 mg/(10.0 mL water 1148 mg/mmol)=498 μM    -   Stock 4× Buffer: 200 mM HEPES, pH 7.5; 400 mM NaCl        The stock solutions were prepared using ACS grade water (Aldrich        32,007-2). 4× buffer was prepared using NaCl 99:999% (Aldrich        20,443-9), HEPES (Gibco-BRL 11344-025), and sodium hydroxide        99.99% (Aldrich 30,657-6), and was treated with Chelex® 100        (BioRad 143-2832) prior to use to remove trace iron        contaminants. These were combined to form the reaction mixture        analyzed by UV-vis spectroscopy: 400 μL 4× Buffer, 200 μL        Ascorbic Acid Stock, 979.9 μL Water, 20.1 μL GdTex Stock.

In brief, a reaction mixture was prepared containing ascorbate, buffer,and water. A lesser amount of texaphyrin catalyst, eg., 0.025 molarequivalent, was added to this reaction mixture, vortexed briefly, andplaced in a quartz cuvette (0.2, 0.5, 1.0, 2.0, 5.0, or 10 mm pathlength as needed to give ca. 1.8 absorbance reading at 266 nm). Thecuvette was placed in a UV-vis spectrophotometer and the absorbance wasread every 60 seconds for ten minutes. The absorbance at 266 nm plottedagainst time initially gaive a straight line. The slope of initial timepoints (eg., plotting as M concentration vs time in minutes) provided arate which was used as a point in a saturation (Michaelis-Menton) plot.This process was repeated at various concentrations of ascorbate. Abackground rate of ascorbate oxidation was also measured at eachconcentration of ascorbate using this procedure without GdTex catalyst.The resulting background rate of oxidation was subtracted from the rateof oxidation in the presence of catalyst. Data was fitted to theMichaelis-Menton equation (initial reaction velocity/catalystconcentration=(k_(cat)×substrate concentration)/(K_(M)+substrateconcentration), where substrate was ascorbate, catalyst was texaphyrincomplex, k_(cat) is the first-order rate constant for the catalyst, andK_(M) is the dissociation (or Michaelis) constant for thecatalyst/substrate complex. For GdTex, k_(cat)=0.35 min-1 and K_(M)=1.06mM.

Results. The results of this analysis are depicted in FIG. 7 whichresults demonstrate that in the presence of GdTex, ascorbate wasoxidized and further that the test compounds have probable radiationsensitization activity.

2B. By following the procedure of Example 2A and substituting motexafingadolinium with motexafin lutetium it was determined that ascorbate wasoxidized in the presence of motexafin lutetium but to a lesser extentthan in the presence of motexafin gadolinium. Thus, taken in conjunctionwith the results of Example 1, probable radiation sensitization activityof motexafin lutetium is associated with a NADPH metabolic pathway,whereas the probable radiation sensitization activity of motexafingadolinium is more strongly associated with an ascorbate metabolicpathway, suggesting that co-administration of both motexafin gadoliniumand motexafin lutetium can combine to more effectively catalyze theproduction of hydrogen peroxide from both pathways.2C. By following the procedure of Example 2A and substituting ironporphyrin for the texaphyrin, it is indicated that ascorbate can besimilarly oxidized and that the test compound will have probableradiation sensitization activity.2D. By following the procedure of Example 2A, using an initial ascorbateconcentration of 1.23 mM and a texaphyrin concentration of 61.5 μM, theresults summarized in below in Table 1 were obtained.

TABLE 1 Ascorbate Depletion Texaphyrin initial rate (V₀) μM/min LuTex−2.33!0.16 ErTex −3.55 DyTex −5.92 GdTex −9.98!1.03 EuTex −9.45!0.91SmTex −10.45!0.50  NdTex −8.85!0.20 MnTex −3.0 (FeTex)₂O −30.6 CoTex−28.9 CdTex −3.4 Di-amino MnTex −3.4 Di-amino GdTex −10.3 Di-amino LuTex−9.7These results eivdence that in the presence of the tested texaphyrins,ascorbate was oxidized, and further that the test compounds haveprobable radiation sensitization activity.

Example 3 Production of H₂O₂ in the Presence of NADPH under ApproximatePhysiologic Conditions.

This example illustrates the measurement of hydrogen peroxide generationfrom the oxidation of NADPH, as an indicator of radiation sensitizationpotential.

3A. Materials and Method.

The following stock solutions were prepared:

-   -   Stock NADPH (Sigma N 7505) (prepared fresh): 10.14 mg/(10        mL×833.4 mg/mmol)=1.217 mM    -   Stock LuTex: 5.02 mg/(10.0 mL water×1166 mg/mmol)=431 M    -   Stock 4× Buffer: 200 mM HEPES, pH 7.5; 400 mM NaCl        These were combined to form the reaction mixture: 200 μL 4×        Buffer, 200 μL NADPH Stock, 371.8 μL Water, 28.2 μL LuTex Stock.        This was analyzed as indicated below. As a control, a solution        in which the LuTex complex was not added was prepared as above,        with suitable adjustment of the volume of water, and analyzed        concurrently. In similar manner, a solution was prepared as        above except that 2 μL catalase (Boeringer-Mannheim 106 836, now        Roche Molecular Biochemicals, Indianapolis, Ind.) was added, and        analyzed concurrently.

A reaction mixture was prepared containing NADPH, buffer, and water.Texaphyrin catalyst, eg., 0.05 molar equivalent, was added to thisreaction mixture, vortexed briefly, and incubated at ambient temperaturein the dark. Aliquots of 50 L were removed every 20 minutes, and addedto a reagent which produces a characteristic color in the presence ofH₂O₂ (Bioxytech® H₂O₂-560™, R&D Systems, Minneapolis, Minn.). A 25 mMsolution of H₂O₂ was prepared by dilution of 30% H₂O₂. The absorbance at240 nm was used to standardize this solution, which was further dilutedand used to construct a standard H₂O₂ curve at 560 nm in conjunctionwith the H₂O₂ colorimetric reagent. Further details on the use of theBioxytech® H₂O₂-560™ kit are available from the package insert.

Measurements of the absorbance at 560 nm (with appropriate subtractionof the background absorbance of the reagent at this wavelength) weremade of samples taken at the various time points.

Results. After conversion of absorbance to H₂O₂, a plot of H₂O₂ vs. timeshowed that H₂O₂ was produced only in the reaction mixture whichcontained LuTex and no catalase. Approximately 40 μM H₂O₂ was producedover the course of 100 minutes. The, results of this example areillustrated in FIG. 8, which demonstrates that in the presence of LuTex,NADPH generates hydrogen peroxide. The test compound has probableradiation sensitization activity.

3B. By following the procedure of Example 3A and substituting redoxcycling agents for the texaphyrin, it is indicated that reactive oxygenspecies can be similarly generated and that the test compound will haveprobable radiation sensitization activity. Suitable redox cycling agentsfor use in this example include, for instance, alloxan, phenazinemethosulfate, menadione, copper/putrescine/pyridine, methylene blue,paraquat, doxorubicin andruthenium(II)tris-(1,10-phenanthroline-5,6-dione). To the extent theseredox cycling agents do not preferentially accumulate in tumors, suchmolecules can be coupled to a compound that does preferentiallyaccumulate in a tumor.

Example 4 Production of H₂O₂ in the Presence of Ascorbate underApproximate Physiologic Conditions

This example illustrates the measurement of hydrogen peroxide generationfrom the oxidation of ascorbate, as an indicator of radiationsensitization potential.

In this example, the following stock solutions were prepared:

-   -   Stock ascorbic acid (Sigma A 5960) (prepared fresh): 9.98 mg/(10        mL×176.12 mg/mmol)=5.67 mM    -   Stock GdTex: 5.76 mg/(10.0 mL water×1148 mg/mmol)=502 μM    -   Stock 4× Buffer: 200 mM HEPES, pH 7.5; 400 mM NaCl        These were combined to form the reaction mixture: 200 μL 4×        Buffer, 39 μL ascorbic acid Stock, 517.3 μL Water, 43.7 μL GdTex        Stock. The reaction mixture was analyzed for hydrogen peroxide        as indicated above. As a control, a solution in which complex        was not added was prepared as above, with suitable adjustment of        the volume of water, and analyzed concurrently.

Measurements of the absorbance at 560 nm (with appropriate subtractionof the background absorbance of the reagent at this wavelength) weremade of samples taken at the various timepoints. It was noted that thecontrol solution gave a uniform background absorbance which was similarat all time points, whereas the mixture containing GdTex gave anincreasing amount of absorbance over time. (Sugars are known to producehigh background absorbance in this assay, see manufacturers insert fordetails.)

Results. After conversion of absorbance to H₂O₂, a plot of H₂O₂ vs. timeshowed that H₂O₂ was produced in the reaction mixture which containedGdTex. The plot (FIG. 9) was linear over the time interval of 75 to 200minutes, with approximately 35 μM H₂O₂ produced over this interval. Thisdemonstrates that in the presence of GdTex, ascorbate generates hydrogenperoxide. The test compound has probable radiation sensitizationactivity.

Example 5 Production of H₂O₂ by the Gadolinium(III) Complex ofTexaphyrin in the Presence of Ascorbate under Cell Culture Conditions

The proliferation of MES-SA human uterine cells (Harker, W. G.;MacKintosh, F. R.; Sikic, B. I. Cancer Res. 1983, 43, 4943-4950) grownin RPMI-1640 medium in the presence of ascorbate or complex was used toassess the formation of hydrogen peroxide under cell culture conditions.

5A. Materials and Methods. MES-SA cells were allowed to adhere to (4)96-well microtiter plates (4000 cells per well) overnight in 160 μL RPMImedium. Stock ascorbate 3.0 mM in medium (80 μL) was serially diluted(1:3) in rows B through F (discarding the final 80 μL). Row G was usedfor no-ascorbate control. Stock solutions of GdTex (2 mM in 5% mannitol)diluted in medium and 5% mannitol were prepared and added to the platesto give a final volume of 200 μL in all wells. Columns 2 and 3 contained100 μM GdTex; columns 4 and 5 contained 75 μM GdTex, columns 6 and 7contained 50 μM GdTex; columns 8 and 9 contained 25 μM GdTex; andcolumns 10 and 11 contained no GdTex (all concentrations final). Finalmannitol concentration was 0.25% in all wells. The plates were incubatedat 37 C under a 5% CO2/95% air atmosphere. Complex-containing medium wasexchanged for fresh medium after 5 hours, and plates were incubated anadditional 72 hours prior to analysis for viability using thetetrazolium dye, MTT (Mosmann, T. J. Immunol. Methods 1983, 65, 55-63).In brief, 20 μL MTT dye (Sigma Chemical, St. Louis, Mo., catalogue no. M2128, 5 mg/mL solution in phosphate buffered saline) was added to cellsin media to give a final volume of 200 μL. The plates were incubated at37° C. for ca. 2 hours, whereupon the media was removed and isopropylalcohol (100 μL/well) was added. Plates were vortexed for ca. 3 minutesto dissolve MTT formazan, then read on a microplate reader at 560-650nm. Plate absorbances were normalized to wells containing neitherascorbate nor GdTex to allow plate to plate comparison. Data for eachconcentration of GdTex and ascorbate is the average of eight wells.

Results. Ascorbate alone had no inhibitory effect at concentrations ator below 333 μM in the absence of GdTex, whereas complete cytotoxicitywas observed at all concentrations of GdTex at this concentration ofascorbate. GdTex had no cytotoxic effect in the absence of ascorbate. Adose-response was observed towards GdTex at 111 μM and 62.5 μMconcentrations of ascorbate.

The results of this analysis are illustrated in FIG. 10. The toxicity ofcells to the combination of sufficient concentrations of GdTex andascorbate is attributed to the production of toxic quantities ofhydrogen peroxide and suggests that motexafin gadolinium has probableradiation sensitization activity.

Example 6 Production of H₂O₂ by the Lutetium(III) Complex of Texaphyrinin the Presence of Ascorbate under Cell Culture Conditions

The proliferation-of MES-SA human uterine cells grown in RPMI-1640medium in the presence of ascorbate and LuTex was used to assess theproduction of hydrogen peroxide under cell culture conditions followingthe protocol outlined in the previous example. Ascorbate alone had noinhibitory effect at concentrations at or below 333 μM in the absence ofLuTex, whereas a dose-response was observed towards LuTex at thisconcentration. No inhibitory effect was seen at lower concentrations ofascorbate. LuTex had no inhibitory effect in the absence of ascorbate.

The results of this analysis are illustrated in FIG. 11. The toxicity ofcells to the combination of sufficient concentrations of LuTex andascorbate is attributed to the production of toxic quantities ofhydrogen peroxide and suggests that LuTex has probable radiationsensitization activity.

Example 7 Production of H₂O₂ by the Lutetium(III) Complex of Texaphyrinin the Presence of NADPH under Cell Culture Conditions

The proliferation of EMT-6 mouse mammary sarcoma (Rockwell, S. C., etal., J. Natl. Cancer Inst. 1972, 49, 735-749) cells grown in RPMI-1640medium in the presence of NADPH and motexafin lutetium was used toassess the production of hydrogen peroxide under cell culture conditionsfollowing the protocol outlined in the previous example. NADPH alone hadno inhibitory effect at concentrations at or below 1000 μM in theabsence of motexafin lutetium, whereas a dose-response was observedtowards motexafin lutetium at this concentration and at 333 μM. Noinhibitory effect was seen at lower concentrations of NADPH. Motexafinlutetium had no inhibitory effect in the absence of NADPH.

The results of this analysis are illustrated in FIG. 12. The toxicity ofcells to the combination of sufficient concentrations of motexafinlutetium and NADPH is attributed to the production of toxic quantitiesof hydrogen peroxide and suggests probable radiation sensitizationactivity for motexafin lutetium.

Example 8 Production of H₂O₂ by the Gadolinium(II) Complex of Texaphyrinin the Presence of NADPH under Cell Culture Conditions

The proliferation of EMT-6 mouse mammary sarcoma (Rockwell, S. C., etal., J. Natl. Cancer Inst. 1972, 49, 735-749) cells grown in RPMI-1640medium in the presence of NADPH and GdTex was used to assess theproduction of hydrogen peroxide under cell culture conditions followingthe protocol outlined in the previous example. NADPH alone had noinhibitory effect at concentrations at or below 1000 μM in the absenceof GdTex, whereas a dose-response was observed towards GdTex at thisconcentration and at 333 μM. No inhibitory effect was seen at lowerconcentrations of NADPH. GdTex had no inhibitory effect in the absenceof NADPH.

The results of this analysis are illustrated in FIG. 13. The toxicity ofcells to the combination of sufficient concentrations of GdTex andNAD(P)H is attributed to the production of toxic quantities of hydrogenperoxide and suggests that motexafin gadolinium has probable radiationsensitization activity, albeit less than that of motexafin lutetium in aNADPH dominated metabolic pathway.

Example 9 The Cytotoxic Effect of L-Buthionine-[S,R]-Sulfoximine (BSO)in the Presence of the Gadolinium(II) Complex of Texaphyrin under CellCulture Conditions

The proliferation of MES-SA human uterine cells grown in McCoys 5Amedium in the presence of a thiol depleting agent (BSO) or GdTex wasused to assess the combined effect of these agents. The protocoloutlined in the previous example was followed, with the followingchanges:

1. The serially diluted compound was 200 μM BSO (Sigma Chemical, St.Louis, Mo., catalogue no. G1404).

2. The cells were allowed to incubate in the presence of the two drugsfor ca. 72 hours, prior to exchange of media and analysis using thecolorimetric (MTT) assay, as described, e.g., in Example 5.

GdTex had an inhibitory effect of ca. 50% in the absence of BSO. Forclarity, the data has been normalized to discount the cytotoxic effectof GdTex alone. BSO alone had an inhibitory effect in the absence ofGdTex of about 10% at the highest (50 μM) concentration tested. In thepresence of increasing amounts of GdTex, an increase in the cytotoxicdose response of the cells towards BSO is seen. This indicates thatcooperative cell growth inhibition occurs in the presence of the twoagents.

The results of this example are illustrated in FIG. 14.

Example 10 The Cytotoxic Effect of the Gadolinium(III) Complex ofTexaphyrin and Ionizing Radiation, with and withoutL-Buthionine-[S,R]-Sulfoximine (BSO) under Cell Culture Conditions

The response of MES-SA cells to ionizing radiation was studied using themicrotiter plate format to confirm radiation sensitization potential.This assay is used to assess rapidly the effects of irradiation under avariety of conditions.

10A. Materials and Methods. MES-SA cells were incubated at 37° C. andallowed to adhere to 96-well microtiter plates (1000 cells per well)overnight in 160 μL McCoys 5A containing 10% FBS (Gibco-BRL) and ca. 2%penicillin/streptomycin solution (Sigma). BSO or media (20 μL) wasadded, respectively to test and control wells, 24 h prior toirradiation. Metallotexaphyrin complex (up to 100 μM) was added to thetest wells to give a final volume of 200 μL, 18-24 h prior toirradiation. Plates were irradiated using a 137 Cs irradiator (Model 40Gammacell, J. L. Shepherd & Assoc., San Fernando, Calif.) at a dose rateof 33.25 Rad/min. A lead brick (2″ diameter) was positioned to protect acolumn of wells (no. 11) from ionizing radiation. Plate absorbances werenormalized to shielded wells to allow plate-to-plate comparison. Controlexperiments demonstrated that data from columns 1-9 were not effected byshielding. Reversing the position of texaphyrin complex-containing wellshad no effect on assay outcome. Medium was exchanged immediately afterirradiation, and plates were incubated an additional 120 h prior toanalysis for viability using the MTT assay (as described, e.g., inExample 5).

Results. Treatment of MES-SA cells with up to 50 μM GdTex for 24 h priorto irradiation had a small effect on apparent viability, afteradjustment for the effect of GdTex alone (FIG. 17A). Radiationsensitization became more significant at the 100 μM GdTex concentration.In the parallel experiment in which all cells were pre-incubated with100 μM BSO, a greater GdTex-dependent decrease in apparent viability wasobserved (FIG. 17B). The effect of BSO appeared to be dose-dependentfrom 0 to 100 μM, and leveled off at higher BSO concentrations (data notshown).

These results corroborate the correlation between radiationsensitization potential and reactive oxygen species production from acellular metabolite having a standard biochemical reduction potentialmore negative than the standard biochemical reduction of oxygen/hydrogenperoxide. Additionally, these results indicate that the radiationsensitization potential of GdTex, and presumably of other reactiveoxygen species-producing sensitizers, can be augmented byco-administration of a thiol-depleting agent or other agents thatinterfere with cellular metabolic pathways.

10B. In corresponding experiments sensitization was not obtained whenLuTex was substituted for GdTex, or when RPMI prepared with dialyzedserum (an ascorbate-free medium) was substituted for McCoys 5A withnormal serum (an ascorbate containing medium).

Example 11 Radiation Sensitization as Determined by Clonogenic Assay

The response of MES-SA cells to ionizing radiation was studied using aclonogenic assay format to confirm radiation sensitization potential.

Materials and Methods. MES-SA cells (200 to 5,000 cells per dish) wereplated in T-25 flasks in 8.5 mL McCoys 5A containing 10% FBS (Gibco-BRL)and ca. 2% penicillin/streptomycin solution (Sigma) and incubated at 37°C. overnight. Stock BSO, ascorbate, GdTex (or a control 5% mannitol)solutions were prepared and 0.5 mL of each added to each flask to give afinal volume of 10 mL (final concentrations of BSO, ascorbate and GdTexwere 100 μM, 5 μM and 50 μM, respectively) and the cells incubated for24 hr, whereupon the flasks were irradiated using a 137 Cs irradiator(Model 40 Gammacell, J. L. Shepherd & Assoc., San Fernando, Calif.) at adose rate of 0.805 Gy/min. Medium was removed immediately afterirradiation, the cells were washed with fresh medium (5.0 mL) followedby the addition of fresh medium (10 mL) and incubation for an additional11 days. Colonies were fixed and stained with 1% crystal violet and thencounted.

Results. As illustrated in FIG. 18, treatment of MES-SA cells witheither 50 μM GdTex or 100 μM BSO for 24 h prior to irradiation had arelatively small effect on clonogenic survival as a function ofradiation. Co-administration of 50 μM GdTex and 100 μM BSO resulted in aradiation response significantly greater than the sum of the responsesobtained from them individually.

These results demonstrate that sensitization of the effects of ionizingradiation by GdTex can be confirmed in a clonogenic assay usingascorbate-containing medium, and that the co-administration of GdTex andBSO synergistically enhances the effects of ionizing radiation.

Example 12 The Cytotoxic Effect of Antimycins A and the Gadolinium(III)Complex of Texaphyrin under Cell Culture Conditions

The proliferation of MES-SA human uterine cells grown in McCoys 5Amedium in the presence of the thiol depleting agent BSO, with varyingconcentrations of a mitochondrial inhibitor (Antimycins A) and GdTex wasused to assess the combined effect of these agents.

Materials and Methods. MES-SA cells were incubated at 37° C. and allowedto adhere to 96-well microtiter plates (2000 cells per well) overnightin 180 μL McCoys 5A containing 10% FBS (Gibco-BRL) and ca. 2%penicillin/streptomycin solution (Sigma). BSO (20 μL) was added to allwells followed by incubation for a further 24 h. The medium wasexchanged and Antimycins A was serially diluted at concentrationsranging from 0 to 20 μM and after 0.5 hr metallotexaphyrin complex (upto 100 μM) was added to the test wells to give a final volume of 200 μL,followed by incubation for 20 h, whereupon the medium was exchanged andthe plates were incubated an additional 52 h prior to analysis forviability using the MTT assay.

Results. In the presence of increasing amounts of GdTex, an increase inthe cytotoxic dose response of the cells towards Antimycins A is seen.This indicates that cooperative cell growth inhibition occurs in thepresence of the two agents. The results of this example are illustratedin FIG. 19.

From the foregoing description, various modifications and changes in thecomposition and method will occur to those skilled in the art. All suchmodifications coming within the scope of the appended claims areintended to be included therein.

1. An injectable formulation comprising motexafin lutetium, water, andacetic acid, wherein the injectable formulation is pharmaceuticallyacceptable to a human and stored in a sealed container that comprises ahead space purged with a gas to exclude oxygen.
 2. The injectableformulation of claim 1, wherein the injectable formulation has a pH ofabout 5.4.
 3. The injectable formulation of claim 1, wherein the sealedcontainer comprises a glass vial.
 4. The injectable formulation of claim1, wherein the gas comprises nitrogen gas.
 5. The injectable formulationof claim 1, further comprising a suitable excipient selected fromlactose, dextrose, sucrose, sorbitol, mannitol, and syrup.